L15 Higher Order Chromatin Regulation And Gene Expression PDF

L15 Higher Order Chromatin Regulation And Gene Expression 5BBG0205 Molecular Basis of Gene Expression Higher order chromatin regulation and gene expression Professor John Strouboulis, Ph.D. Chair in Molecula...

L15 Higher Order Chromatin Regulation And Gene Expression 5BBG0205 Molecular Basis of Gene Expression Higher order chromatin regulation and gene expression Professor John Strouboulis, Ph.D. Chair in Molecular Erythropoiesis [email protected] December 6, 2023 Outline of lecture 1) Interphase chromosome structure 2) DNA elements that direct gene transcription 3) Methods for capturing enhancer and silencers genome-wide  Learning objectives Explain the structure and nuclear positioning of interphase chromosomes. Compare the types of DNA elements that influence gene transcription. Identify the characteristics of enhancers, silencers and insulators. Describe methods to measure enhancer and silencers. Higher order chromatin regulation Part 1 and gene expression Interphase chromosome structure 5BBG0205 Molecular Basis of Gene Expression 2  The human diploid genome has roughly 6 megabases of DNA Karyotype of the human genome Chromosome compaction Beads-on-a-string Chromatin: DNA and proteins packaged into chromosomes In interphase, chromosomes slightly less compacted than traditional X shaped chromosomes. In interphase: o DNA wraps around nucleosome 1.65 times, completed with H1 histones. o Folds up to form 30nm fibers which form 300nm loops. o 300nm loops compressed to form 250nm fiber. o Coiling of 250nm fiber produces chromatid 3  Chromosome compaction M phase Chromatin: DNA and proteins packaged into chromosomes Only looks like X shape during mitosis. What is an interphase of the cell cycle? G1 S G2 Interphase is preparation phase for mitosis During interphase, the cell grows (G1), replicates its DNA (S) Chromosomes decondensed in G1 S and G2 phases, condense in mitosis and decondense again during interphase. 4  Chromosome compaction Interphase Chromatin: DNA and proteins packaged into chromosomes What are chromosome territories? Chromosomes exist in a decondensed state at interphase. Territories are typically 1 to 2 microns in diameter. There is limited overlap between territories (but some intermingling at the borders). Transcriptionally inactive regions are in the dense interior of the territory. Transcriptionally active regions are more decondensed and localised near the periphery of the territory. Chromosome positioning is not random. Adjacent chromosomes at greater risk of chromosomal translocations (some types of cancer). At the borders of each chromosome territory there is some intermingling. Chromosomes occupy discreet territories in the nucleus. Transcriptionally active regions tend to be towards the outside of the territory and can share transcriptional machinery. 5  DNA probes are specific to each chromosome and have different coloured fluorochromes. Chromosome territory positions in the nucleus Radial chromosome positioning Preferred chromosome neighbouring 19 18 Cell type specific Cell type independent DNA FISH using whole chromosome paints Chr 18 –gene poor Chr 19 –gene rich Croft et al 1999 J Cell Biol 145: 1119-1131 Gene dense chromosomes tend to localise towards the inside of the nucleus as this is where the majority of the transcriptional machinery lies. Less gene-dense chromosomes will be located towards the periphery of the nucleus. Heterochromatin (closed chromatin) tends to be towards the periphery of the nucleus. 6  Chromatin Compartmentalization is an Organizing principle of the nucleus B compartments are localized Chromosome to the nuclear periphery territories A compartments are more interior Euchromatin active Heterochromatin Inactive Active and Inactive Genomic Regions in the Eukaryotic Nucleus Chromosome territories also have territories within them. A compartment = associated with transcription, loop wards interior of nucleus. A alternates with B. B is heterochromatinised. Can individual genes in the INTERPHASE nucleus be visualized and topographically analyzed? How can we do that? Recent advances in fluorescence in situ hybridization (FISH) have allowed individual genes in the interphase nucleus to be visualized and topographically analyzed. Green: single transgene Red: heterochromatin (e.g. centromeres; highly condensed, inactive) Chromosome compartments/domains depend on the stage of mitosis the chromosome is at 7  Summary from Part 1 Levels of DNA Compaction Double-stranded DNA wraps around histone proteins to form nucleosomes that have the appearance of “beads on a string.” The nucleosomes are coiled into a 30-nm chromatin fiber. When a cell undergoes mitosis, the chromosomes condense even further. Interphase chromosomes occupy discrete chromosome territories in the nucleus. Summary from Part 1: Levels of genome organization 8  Higher order chromatin regulation Part 2 and gene expression DNA elements that direct gene transcription 5BBG0205 Molecular Basis of Gene Expression Hierarchies of functional chromosome structure in gene regulation Nucleosome Supranucleosomal Nuclear scale scale scale DNA sequence itself is the lowest level of control of gene regulation. 9  What controls gene transcription? P Gene Transcription Transcription Preinitiation complex (PIC) factor factor RNA polymerase II Genes contains core regulatory elements e.g. core promotor where PIC binds. Other regulatory elements can be upstream or downstream. Promoter sequences Proximal promotor element serves as a landing platform for RNA polymerase or initiation or elongation factors. 10  Promoter sequences Gene regulatory sequences can bind other regulatory sequences and make contact with the promoter to stimulate transcription. Distal regulatory elements Schaffner, 2015 Biol. Chem. Enhancers —DNA sequences that can increase the transcription rate of a gene Silencers —DNA sequences that can decrease the transcription rate Insulators —DNA sequences that can block these effects They can be upstream, downstream or directly inside the gene and can bind promotors, transcription factors, cofactors or chromatin remodelling enzymes. Silencers block transcription initiation. Insulators provide a wall that block binding between an enhancer and promoter. AKA boundary elements. 11  Enhancers -DNA elements that increase the rate of transcription from a gene promoter. -First identified in SV40, a small virus that grows in monkey cell lines (1981) -Soon after, cellular enhancers were identified (1983) (Immunoglobulin heavy chain gene) -Sequences that, when cloned into a plasmid next to a human beta-globin gene with a weak promoter, increased transcription -Different from promoters because: they function in either orientation they function either upstream or downstream of the gene reverse orientation beta-globin gene weak promoter “enhancer' - + +++ +++ +++ Experiments to test enhancer function Epigenetic signature: histone tail acetylation, histone H3-lysine 4 monomethylation (H3K4me3) If you add enhancers there is a significant increase in transcription. Still activates transcription regardless of positioning and orientation. They have a specific epigenetic signature: histone tail acetylation, histone H3 lysine 4 monomethylation. (H3Kame3). Enhancers Enhancers are typically composed of a cluster of transcription factor binding sites that work cooperatively to enhance transcription The transcription factors that bind to enhancers are called transcriptional activators From Spitz and Furlong, 2012 Nature TFs bind to specific motifs that they recognise. They will acetylate or methylate the nucleosomes to loosen the chromatin and allow transcription. 12  What do enhancers do? Cell type ”A” E E E P Gene E E Cell type ”B” E –enhancer P –promoter Across the genome there are hundreds of thousands of enhancers, and some genes are regulated by combinations of multiple enhancers. Some enhancers act upon their gene targets under restricted circumstances (specific cell type, developmental stage, extracellular signalling cues). They function to fine-tune gene expression at different developmental stages, in different cell types and in response to different signalling cues. Tissue specific genes may be regulated by combinations of multiple enhancers. Enhancers can fine tune gene expression. Where are enhancers located? E E P Exon E Exon Exon E Sometimes within a few kilobases of the gene. Can be intronic. Can be either upstream or downstream of the gene. Can be hundreds of kilobases from the gene (and even megabases away). Enhancer may be in a gene that is not linked to the gene of interest 13  What controls gene transcription? Histone modifying enzyme Events involved in gene transcription Chromatin structure and gene expression Organization of chromatin into the tightly condensed 30- nm fibre and 'beads-on-a-string' 10-nm fibre. nascent RNA Region of DNA containing an actively transcribed gene. The transcribed regions also contain DNAseI Hypersensitive Sites (HS), which may be associated with a small stretch of DNA devoid of nucleosomes or simply reflect a different nucleosomal structure (positioning or histone modification). Regions of general DNAseI hypersensitivity sensitivity correlate with acetylation of the histone tails, defining a structural and functional 'domain' for gene activity that is characteristic of 'open' chromatin. Francastel C., et al., 2000 Nature review Chromatin that is looser is more sensitive to DNAse 1 than tightly bound chromatin. 14  The Locus Control Region (LCR): a super enhancer for the β-globin gene locus Arrows: DNAse I Hypersensitive (HS) sites Genes become activated and silence depending on developmental stage and age. Contains DNAse 1 hypersensitive sites. Suggests that there is a super enhancer here (many enhancers in one site). This can significantly increase the rate of expression. This is a feature of certain genes that need to drive very high levels of expression e.g. lots of haemoglobin needed for red cells in this case. Beta globin genes code for beta chain of haemoglobin. 15  A long-range enhancer Different regulatory elements may drive Sonic hedgehog (Shh) gene is regulated by distal transcription in different scenarios enhancers, including one that is active only in -cell type specificity developing limb buds, and embedded in an -developmental stages intron of another, distant gene (Lmbr1). -cell cycle control -response to stimuli Expression pattern of the Sonic hedgehog gene, shown by fusing its regulatory elements Hindbrain and to a LacZ reporter gene notochord-specific enhancers Limb bud-specific enhancer gut-specific enhancers Lettice, 2003 Hum. Mol. Genet. Shh has enhancers within other genes and closer. Different sets of enhancers can induce the expression of different kinds of tissue e.g. different enhancers for limb bud, gut, hind brain, spinal cord. Silencers -DNA elements that decrease the rate of transcription from a gene promoter. -Less well studied than enhancers -Different from promoters because: they function in either orientation they function either upstream or downstream of the gene reverse orientation beta-globin gene strong promoter “silencer' - +++ - - - -Can silence transcription of genes that are positioned at large distances away. -Epigenetic signature largely unexplored, but likely to involve histone 3 Lysine 27 trimethylation (H3k27me3) Polycomb binding ??? Function in any orientation or location. 16  Insulators -DNA elements that block communication between DNA elements or separate defined chromatin regions Insulators -These DNA elements contain a sequence motif of CCGCGNGGNGGCAG (very GC-rich) -These motifs bind a transcription factor called CTCF (CCCTC binding factor) -CTCF contains 11 zinc fingers that enables it to bind to DNA and also to homodimerize CTCF -CTCF binding can delineate domains of gene activity 11 zinc fingers allows CTCF to bind to many GC rich regions and to homodimerize. CTCF can organise domains of activity or inactivity. 17  Insulators -CTCF binds to chromatin often in conjunction with the cohesin complex -cohesin forms a ring-like structure Cohesin complex Cohesin can help ‘pull out’ loops of DNA (loop extrusion). Genes/regulatory elements that are in the same loop domain can be transcribed at the same time. CTCF and cohesin help topologically organise domains and allow DNA looping where promoters and enhancers loop together. 18  Higher order chromatin regulation Part 3 and gene expression Methods for capturing enhancers and silencers genome-wide 5BBG0205 Molecular Basis of Gene Expression How can we measure the complex organization of chromatin interactions? ChIP-seq 3C robust method for studying protein– capable of analyzing long range chromatin DNA interactions coupled to Next interactions in nuclear space (the data Generation Sequencing (NGS) for interpretation is complicated) genome-wide identification of transcription factor binding sites. 3C has limited detection scope only provides linear information of protein binding sites along chromosomes. ChIP-seq – measures specific but linear information. Does not tell you about 3-dimensional orientation 3C – provides 3-dimensional information. 19  Chromatin immunoprecipitation sequencing (ChIP-seq) POI = Protein of interest (could be a transcription factor or a histone modification) Formaldehyde introduces cross links between DNA and bound proteins, so it “freezes” the binding at the time of interest. Antibody specific to transcription factor of interest, which will be bound to DNA. Map where the TF binds. ChIPseq data visualised across the mouse beta-globin locus GATA1 FOG-1 cohesin CTCF 3’ HS1 LCR HS60/62 βmin βmaj βh1 εy 20  Peaks of binding in locus control region. Chromosome Conformation Technologies Resulting 3C is a line of DNA that are in contact with each other. 21  Histone modification ChIPseq for marking promoters and enhancers, ATAC-seq for accessible chromatin etc. Chromosome conformation capture: principle of how it works 3C 4C 5C 22  Conformation Technologies Biological impact of Chromosome 3C methods have led to a number of biological insights, including the discovery of new structural features of chromosomes, the cataloguing of chromatin loops, and increased understanding of transcriptional regulation mechanisms (the disruption of which can lead to disease). 3C methods have demonstrated the importance of spatial proximity of regulatory elements to the genes that they regulate. For example, in tissues that express globin genes, the β-globin locus control region forms a loop with these genes. This loop is not found in tissues where the gene is not expressed. This technology has further aided the genetic and epigenetic study of chromosomes both in model organisms and in humans. These methods have revealed large-scale organization of the genome into topologically associating domains (TADs), which correlate with epigenetic markers. Some TADs are transcriptionally active, while others are repressed. Moreover, CTCF and cohesin play important roles in determining TADs and enhancer-promoter interactions. The result shows that the orientation of CTCF binding motifs in an enhancer-promoter loop should be facing to each other in order for the enhancer to find its correct target. Chromatin loops will vary between tissues and developmental stages. 23